The present invention relates generally to the art of separating charged molecular species, and, in particular, to new devices, systems and methods for practicing same.
Capillary gel electrophoresis is one of the most widely used separation techniques in the biologically related sciences. Charged molecular species such as proteins, peptides, nucleic acids, amino acids and oligonucleotides are separated by causing the species to migrate in a buffer medium under the influence of an electric field. The buffer medium normally is used in conjunction with a low to moderate concentration of an appropriate gelling agent, such as for example, agarose or cross-linked polyacrylamide, to promote the separation and to minimize the occurrence of mixing of the species being separated.
Until recently, electrophoretic separations were conducted in gel slabs or open gel beds which were typically fabricated of agarose or cross-linked polyacrylamide material. More recently, capillary gel electrophoresis techniques combined with photometric detection methods have allowed the automation and rapid quantitative analysis of charged molecules. Furthermore, capillary gel electrophoresis can provide quantitative information about a sample using very small amounts of the sample, gel and buffer relative to traditional slab gel processes. Moreover, high resolution separation of molecules having different effective charges have been achieved by applying electrophoretic principles to polymer solution-filled or gel-filled narrow capillary tubes.
A problem encountered with such separation methods is the compatibility of a particular separation medium with the surface of the separation device. In particular, electro-osmotic flow of a sample to be separated between the separation medium and the inner surface of the separation device occurs when a separation medium is unable to adhere, i.e., adsorb, to the surface of the separation device. Such electro-osmotic flow provides unreliable separation results because the sample flows around the separation medium instead of through it.
Traditional methods aimed at preventing such electro-osmosis include introducing a compound which binds to the inner surface of a capillary tube wall, as well as, to the separation medium prior to injecting the separation medium into the tube. For example, U.S. Pat. No. 5,447,617 to Shieh describes covalently bonding polybutadiene to the inner surface of a capillary tube, introducing polyacrylamide therein and co-polymerizing the polyacrylamide with the polybutadiene. Such precoating techniques, however, are time consuming, inconvenient and costly.
A further problem of conventional capillary gel electrophoresis is encountered with the use of polyacrylamide-based separation media. Such media are injected into the capillary tube in unpolymerized form. Polymerization of the polyacrylamide is then induced within the capillary tube by any number of methods including ultraviolet radiation and chemical catalysts. Such methods are characterized by a lack of uniformity in the pore size distribution of the polymer network formed, and by incomplete polymerization.
Accordingly, attempts have been made to use nonpolymerized separation media for capillary gel electrophoresis. See for example, U.S. Pat. Nos. 5,126,021, 5,468,365 and 5,213,669 which describe separation media which form dynamic entanglements, associations, or cross-links for electrophoretically separating biological samples including protein, DNA and RNA. These separation media are not ideal for separating molecular samples because they are difficult to manipulate, require precoating of the surface of the separation device and/or are not universally compatible with the surface of such separation devices, i.e., do not have temperature-dependant hydrophobic and hydrophilic segments, respectively.
Glass and silica surfaces are most commonly used in conjunction with the separation media described above. For example, capillary columns used in capillary gel electrophoresis are fabricated from lengths of fused silica tubing having diameters on the order of 25 .mu.m to 200 .mu.m and lengths from about 30 cm to about 200 cm. The buffer and gel separation media are pumped directly into the column interiors and electrophoretic techniques are used to separate charged molecular species.
Similarly, such molecular separations have been attempted on the surface of silicon wafers. For example, U.S. Pat. No. 4,908,112 to Pace, hereby incorporated by reference, discloses an analytical separation device in which a capillary sized, i.e., micro-machined conduit is formed by a channel in a semiconductor device. The surface of such a device is conditioned to accept traditional separation media. In particular, the surface of the semiconductor device is thermally oxidized to form a SiO.sub.2 layer. The conduit is then filled with a traditional electrophoretic gel preparation fluid, such as for example, a monomer and a cross-linker of a polyacrylamide.
A major drawback to the use of such a device, however, is the inability of many separation media to adhere/adsorb to the inner wall of the conduit which, as set forth above, creates electro-osmotic flow of a sample between the surface of the gel and the wall of the conduit when an electric field is applied during electrophoresis. When such electro-osmotic flow of the sample occurs, a satisfactory separation of the constituent parts of the sample cannot be obtained.
Thus, attempts have been made to find separation media capable of high adsorbency onto, e.g., silica surfaces, and which are still able to electrophoretically separate molecular species. In particular, the adsorption of nonionic block copolymers of the Pluronic PE type, i.e., poly(oxyethylene)-poly(oxypropylene)-poly(oxyethylene), at hydrophobic silica surfaces have been investigated. See for example, Teberg, F.; Malmsten, M.; Linse, P.; Lindman, B. Langmuir, 1991, 7, 2723-2730 and Malmsten, M.; Linse, P.; Cosgrove, T. Macromolecules, 1992, 25, 2474-2481. The utility of such media, however, is limited by the requirement that the silica surfaces of the separation device be rendered hydrophilic for effective adsorbency thereon by the oxyethylene (E) block of the separation media.
Accordingly, it would be desirable to provide a device for electrophoretic molecular separation that is adapted to accept a molecular separation medium which efficiently and effectively separates molecular species without the problems associated with the above-referenced citations. It would also be desirable to provide a separation medium with the ability to adsorb to a variety of substrate surfaces and to change between liquid and gel-like states for efficient application and removal of the medium from such a molecular separation device. In particular, it would be desirable to provide a device for molecular separation which has a surface with hydrophobic properties which surface can be modified to accept such a molecular separation medium through successive oxidation and protophilic reactions. It would also be desirable to provide methods for rendering hydrophilic surfaces, such as silicon surfaces, competent to receive molecular separation media.